Formaldehyde is a widely used high production chemical that is also released as a byproduct of combustion and off-gassing of various building products. Formaldehyde has been classified as an animal and human carcinogen by the International Agency for Research on Cancer, causing nasopharyngeal cancer. Formaldehyde is also reported to be associated with the induction of leukemia. Formaldehyde is highly reactive with DNA and proteins to induce diverse lesions, with the formation of DNA-protein cross-links (DPC) as the primary genotoxic effect. Internal exposure monitoring and cancer risk assessment of formaldehyde have been challenging due to the lack of suitable biomarkers. This also limits our capability of examining formaldehyde distant effects in organs remote to nasal epithelium, which remains highly controversial. Recently, we have established N2-hydroxymethyl-deoxyguanosine as the first formaldehyde-specific DNA adduct biomarker, and the rich dataset generated using this biomarker is widely used in risk assessment of formaldehyde. However, DPC, formed at high abundance, are biologically more important because they significantly interfere with DNA repair as extremely bulky DNA lesions. However, no biomarkers have been established to quantify formaldehyde-induced DPC due to substantial technical challenges. Previous methods of measuring DPC either utilized non-chemical-specific methods with low sensitivity or relied on radioactive formaldehyde for exposure. The objective of this application is to develop biomarkers and sensitive mass spectrometry-based methods to measure formaldehyde-induced DPC. The central hypothesis is that cysteine-containing cross-links can be developed as biomarkers to quantify formaldehyde-induced DPC, and that proteins cross-linked with DNA can also serve as biomarkers to assess the formation of DPC. This hypothesis has been formulated on the basis of preliminary data produced in the applicant's laboratory and our experience on developing mass spectrometry assays for DNA lesions. We will test the hypothesis by first optimizing enzymatic hydrolysis approaches to digest DPC into small cross-links for mass spectrometry detection, and then developing sensitive mass spectrometry assays to quantify cysteine-containing cross-links. We will also isolate, identify and quantify proteins cross-linked with DNA following formaldehyde exposure as biomarkers of DPC. The approach is innovative because of the novel application of highly sensitive mass spectrometry to develop new formaldehyde-specific biomarkers to quantify DPC formation. The proposed research is significant, because it is expected to establish a set of novel biomarkers of formaldehyde exposure to measure formaldehyde- induced DPC, a current void in formaldehyde biomarker research. Results from this study will also lay a foundation for future studies aiming at establishing molecular dosimetry using these novel biomarkers of DPC to better understand formaldehyde carcinogenicity and its cancer risk assessment.